The opening of the pore in the transmembrane regions of the skeletal muscle Ca2+ release channel or ryanodine receptor (RYR1) is regulated by the binding of a variety of modulators to its cytoplasmic domains. Mutations that produce the two human diseases, central core disease (CCD) and malignant hyperthermia (MH), are also found frequently in cytoplasmic domains. The overall objective of this application is the elucidation of the molecular mechanisms by which binding of cytoplasmic modulators or the presence of mutations alter channel gating. The working hypothesis for this application is that the modulators bind close to and modulate infra or intersubunit contact sites that allosterically regulate the gating of the pore in the transmembrane domain. We also propose that the two cytoplasmic domains where the malignant hyperthermia and central core disease mutations occur interact to form an infra or intersubunit contact site close to the FKBP12 binding site and that mutations in either interacting partner alter both modulator binding and intersubunit communication. The specific aims are to: 1) delineate differences between the channel in the open and closed conformations to identify structural steps in channel gating, 2) assess the contributions of modulators to RYR1 structure, 3) localize the MH/CCD mutation sites and cysteine 3635 in the 3D structure of RYR1 and confirm protein-protein interactions. Structural information will be obtained using cryoelectron microscopy and computer reconstruction. Structural studies will be paralleled with functional analysis using single channel analysis of channels reconstituted into planar lipid bilayers and analysis of [3H]ryanodine binding. Interacting domains within RYR1 will be identified by assessing the ability of expressed fragments to interact using a biosensor assay and by identifying interacting regions using the yeast interaction trap.